The neuroendocrine system programs development through release of steroid and peptide hormones at critical times during the life history of the organism. We are investigating the neuroendocrine basis of an innate behavioral sequence in model insects. At the end of each developmental stage, release of peptide ecdysis triggering hormones (ETH) programs a sequence of respiratory and behavioral events culminating in cuticle shedding and transition to the next stage. The scheduling of these events occurs via a downstream peptide signaling cascade in the central nervous system (CNS). The long-term objectives of this proposal are to gain new insights into how this signaling cascade coordinates the timing and proper sequence of events necessary for survival of the organism. The first objective of the proposal is to elucidate functions for ETHR-A and ETHR-B neurons in the ecdysis sequence by RNA silencing or overexpression of each receptor subtype to distinguish their functions. The second specific aim will define precise functions of peptidergic signaling in initiation and scheduling of the ecdysis behavioral sequence. We will use molecular tools to activate or ablate specific peptidergic ensembles to test their necessary roles in the ecdysis sequence in Drosophila, Aedes, and Bombyx. The third specific aim is to elucidate how the ETH-activated central peptide signaling cascade mediates respiratory inflation. In particular, the critical roles of peptide kinin signaling will be examined using cell-specific ablation, RNA gene silencing and cell-specific activation with light or temperature-activated ion channels. We will use several strategies for hypothesis-testing of the roles of specific peptidergic ensembles in ETH-induced behaviors. These are: 1) correlation of cellular activity and peptide action with behaviors by imaging and electrophysiology, 2) driving ensembles using overexpression of ETH receptor subtypes and cell-specific activation with appropriate ligands (ETH) or channel rhodopsins, and 3) removal of neurons and/or genes by cell-specific ablation and neuropeptide-specific RNA silencing. The ecdysis sequence is a series of clearly defined steps under the control of peptide hormones and transmitters. It provides a favorable model for molecular analysis of a complex developmental process involving respiratory dynamics and behaviors. It is likely that scheduling of these events by peptidergic signaling cascades in the CNS is of general significance and relevance to all animals. Understanding the regulatory mechanisms, which underlie these precisely timed developmental processes will offer new insights into how behavior is controlled by the neuroendocrine system, and may lead to a better understanding of normal and pathological states in humans. Results of these studies also may provide basic new information about vector-borne human diseases involving mosquitoes, flies, ticks, and other arthropods. PUBLIC HEALTH RELEVANCE The ecdysis sequence is a series of clearly defined steps under the control of peptide hormones and transmitters. It provides a favorable model for molecular analysis of a complex developmental process involving respiratory dynamics and behaviors. It is likely that scheduling of these events by peptidergic signaling cascades in the CNS is of general significance and relevance to all animals. Understanding the regulatory mechanisms, which underlie these precisely timed developmental processes, will offer new insights into how behavior is controlled by the neuroendocrine system, and may lead to a better understanding of normal and pathological states in humans. Results of these studies also may provide basic new information about vector-borne human diseases involving mosquitoes, flies, ticks, and other arthropods.